Analysis of isomers in TIMS-Q-q-tof mass spectrometers

ABSTRACT

The invention relates to methods for the detailed analysis of ion mixtures from complex mixtures of organic substances in time-of-flight mass spectrometers which are equipped with a trapped ion mobility spectrometer, a quadrupole mass selector and a fragmentation cell. The invention proposes to analyze ion signals of a first mass mobility map, fragment ion spectra and the identifications of the associated substances as to whether ion mixtures not resolved according to mass and mobility, for example from isomers or isobars, are possibly present, and to subsequently measure the ion signals of interest with method parameters which allow the ion species to be measured separately by means of high mobility resolution.

BACKGROUND OF THE INVENTION Field of the Invention

The invention relates to methods for the mass spectrometric analysis ofcomplex substance mixtures and in particular for the identification ofisomers and isobars by means of time-of-flight mass spectrometers whichare equipped with a trapped ion mobility spectrometer, a mass filter anda fragmentation cell.

Description of the Related Art

The U.S. Pat. No. 7,838,826 B1 (M. A. Park, 2008) discloses a trappedion mobility spectrometer (TIMS), which is also called a trapped ionmobility separator hereinafter. In a trapped ion mobility spectrometer,ions inside a multipolar ion guide system are typically driven through agas stream against an electric field barrier, which preferably has aramp with increasing electric field strength. The ions gather along theramp at positions where the gas friction for one ion species is inequilibrium with the restoring electric force in each case. The ionstherefore gather, spatially separated according to their ion mobility.When sufficient ions have been stored, after a predeterminedaccumulation time, for example, the counter-acting electric field of theelectric field barrier is lowered in a “scan”, which causes the ions tobe released sequentially from lower to higher mobility over the end ofthe ramp and to be transferred to a mass spectrometer, preferably atime-of-flight mass spectrometer with orthogonal ion injection.

FIG. 1 shows a schematic representation of a typical trapped ionmobility separator and an operating mode of the trapped ion mobilityseparator. Gas (7) with entrained ions (6) enters the first vacuum stageof a mass spectrometer through a capillary (8). The ions (6) are pushedinto an RF ion funnel (10) by an electrode (9) and guided to the tube(11) of the trapped ion mobility separator, which comprises segmentedring electrodes. An RF quadrupole field is generated in the tube (11),which keeps the ions close to the axis of the tube (11), by applying RFpotentials with alternating phase to the quadrants (1, 2, 3, 4) of thering electrodes. Moreover, an electric field E(z) having a ramp alongwhich the electric field strength increases (between positions 20 and23), and a flat top (between positions 23 and 24) is generated in thetube (11) by DC potentials at the ring electrodes. A gas flow (14, 16)with a laminar velocity profile pushes the ions towards the ramp. Ionswith the same mobility gather at a position on the ramp where the gasfriction is in equilibrium with the restoring force of the electricfield. After a certain accumulation time (Diagram A), the DC voltagesare decreased (Diagram B), and the ions can leave the tube (11) throughthe diaphragm (13) across the flat top in the sequence of theirmobility.

The lowering of the counter-acting electric field (scan) typically takesbetween 20 and 250 milliseconds. The trapped ion mobility separator canbe very compact. The tube (11) as the main part can have a length ofonly five centimeters, for example.

This type of mobility spectrometer is unique in that the mobilityresolving power depends on the scan speed. FIG. 2 shows the mobilityresolution as a function of the scan duration. For ions with a mobilityof around K₀=0.5, a mobility resolution of R_(mob)=K₀/ΔK₀=60 is achievedwith a scan duration of only 20 milliseconds; with a scan duration of300 milliseconds, the mobility resolution increases to R_(mob)=120.

The U.S. patent application Ser. No. 14/931,163 (M. A. Park et al.;“Acquisition of Fragment Ion Mass Spectra of Ions Separated by theirMobility”), which is incorporated herein by reference, describes amethod to identify organic substances in a complex substance mixture,such as the enzymatic digest of a proteome. The method disclosed thereis preferably conducted with a mass spectrometric system which isschematically depicted in FIG. 3 and comprises the following components:a liquid chromatograph (optional), an ion source, an (accumulative)trapped ion mobility separator, a mass filter, a fragmentation cell anda time-of-flight mass spectrometer with orthogonal ion injection andreflector.

The substances dissolved in a liquid are fed to an ion source,preferably an electrospray ion source, and ionized. Depending on thecomplexity of the substance mixture, the substances may already bechromatographically separated or may originate in unseparated form froma syringe pump.

First a mass-mobility map, as shown schematically in FIG. 4, is acquiredwithout using the mass filter and the fragmentation cell. Typically, theions stored in the trapped ion mobility separator are separatedaccording to their mobility in a mobility scan lasting around 50milliseconds and transferred to the time-of-flight spectrometer, wherethe ion species, separated according to their mobility, are massspectrometrically analyzed. A modern time-of-flight mass spectrometercan acquire 10,000 mass spectra per second, and it can be expedient tosum some of the acquired mass spectra in order to achieve a sufficientlygood quality and a large dynamic range of signals.

For complex substance mixtures, the mass-mobility map comprises a largenumber of ion signals, which typically extend along the mobility axisfor around one to three milliseconds. The mobility resolution here ismuch smaller than the mass resolution. The mass of the ion signalsdecreases along the mobility axis, and singly and multiply charged ionsform strongly dispersed, partially overlapping bands (see FIG. 4). FIG.4 is greatly simplified; on the one hand, it does not show the isotopicdistributions of each ion signal, and on the other, the figure containsonly relatively few ion signals—there can be thousands of ion signalsfor complex substance mixtures.

A first group of ion signals is then selected from the mass-mobilitymap. This group contains a number of ion signals which are arranged intemporal sequence, i.e. they do not overlap in the direction of themobility axis. FIG. 4 shows a possible selection of a group of ionsignals, which are encircled by ellipses in the mass-mobility map.

After the acquisition of the mass-mobility map and the selection of afirst group of ion signals, a mobility scan is subsequently carried outto identify the substances of the ion signals of the group. The massfilter, the fragmentation cell and the time-of-flight mass spectrometerare needed for this purpose. After the trapped ion mobility separatorhas been filled and the mobility scan has started, the mass filter,usually a quadrupole mass filter, is switched to allow ions of the firstion signal from the group to pass through. It is preferable for all ionsof the isotopic distribution to be included here in order to obtainfragment ion spectra whose isotopic distributions agree with those ofreference spectra from spectral libraries. The mass filter is thereforeset to a transmission window of a few daltons. The ions of the first ionsignal thus selected according to mobility (scan time) and mass arefragmented in the fragmentation cell; the fragment ions are measured inthe time-of-flight mass spectrometer as a fragment ion spectrum. As arule, the substance can be identified from this fragment ion spectrum,usually via comparisons with reference spectra of a spectral library. Inthe further course of the same mobility scan, the ions of further ionsignals are selected according to mobility and mass in the same way andfragmented. The time intervals between the ions of the group are chosensuch that the mass filter can be switched to the mass of the ions of thesubsequent ion signal. The further substances of the selected ionsignals of this group are identified by acquiring the correspondingfragment ion spectra. The trapped ion mobility separator is then filledwith a further batch of ions, and the identification of a second groupof ion signals from the mass-mobility map can start.

If the mobility scan takes 20 milliseconds, for example, and an ionsignal typically lasts 2.5 milliseconds on leaving the trapped ionmobility separator, then up to eight signals can be selected for eachgroup in a single mobility scan; for a mobility scan lasting 50milliseconds, each group can comprise around 20 signals. If amass-mobility map comprises around 200 ion signals which are reasonablyuniformly distributed, they can be split up into around 10 groups with ascan lasting 50 milliseconds. If they are not distributed uniformly, itmay be necessary to form more than 10 groups in order to cover allsignals of the mass-mobility map.

Several mass-mobility maps can be acquired and summed to improve thequality. It may furthermore be expedient, for larger molecules, to onlyselect ion signals of ions which are at least doubly charged, since theycan be fragmented more easily than singly charged ions and with a highyield.

Assuming a scan duration of 50 milliseconds each, around 20 of thesemeasuring cycles can be completed, until after around one second a newmass-mobility map is measured, in order to take account of a changeduring a chromatographic separation, for example. Theoretically, around400 ion species per second can be identified in this way. In reality,this number of measurements is not completely achieved, even when somany ion species are present. Even in very complex substance mixtures,it is usually not the case that so many ion species are available,however. The excess measuring capacity can then be used to measure weakion signals in successive measurement cycles several times and to sumthe fragment ion spectra in order to achieve a better quality of spectraand thus a more certain identification.

This method can be modified in a variety of ways depending on theanalytical requirements. A higher mobility resolution can be achieved bylonger scan durations, while at the same time larger numbers of ionsignals can be selected for each group. All ions of an ion source can beutilized by a trapped ion mobility separator with parallel accumulationof ions (U.S. patent application Ser. No. 14/614,456; “Trapping IonMobility Spectrometer with Parallel Accumulation”, M. A. Park and M.Schubert) without ion losses occurring during the mobility scan. FIG. 5illustrates a preferred operating mode of a trapped ion mobilityseparator with parallel accumulation. The extended tube (11) issubdivided into two regions (11 a) and (11 b) here. Ramps withincreasing field strength can be set up in both regions, as can be seenin Diagrams D and E. The first region serves to accumulate ions, whilethe ions of the second region are being subjected to a mobility scan.When the mobility scan is finished, the ions of the first region can betransferred into the second region in around one millisecond.

“Isomers” are molecules which have the same elemental composition, i.e.the same molecular formula, but different primary structures(“structural isomers”) or different secondary or tertiary structures(“conformational isomers”). Isomers have precisely the same mass.“Isobars” are different molecules which are composed of differentelements such that their mass has the same number of atomic mass units(daltons), but not exactly the same mass. Isobars are then said to havethe “same nominal mass”. Molecular ions always have isotopicdistributions which span several daltons. If the molecular ions of twomolecules whose masses differ by only one, two or three daltonsinterfere with each other, it is not possible to separate the isotopicdistributions of these molecules from each other with a mass filter.These molecules are called “near isobars” here. When the term “isomers”is used here, the expression is often to include the “isobars” or “nearisobars” as well.

There is a need to have methods for analyzing complex substance mixtureswhich can be used to identify isomeric and isobaric organic substancesin complex substance mixtures, in particular pharmacological substancesof particular analytical interest, and to determine the mobility K₀ orthe collision cross-section a of the substances as accurately aspossible.

SUMMARY OF THE INVENTION

Experience has shown that not all ion signals of a mass-mobility mapbelong to single homogeneous ion species. Each ion signal can containone ion species, but can occasionally also contain several isomers orisobars which are not resolved according to their mobility, and whoseisotopic distributions overlap. Ion mixtures with sufficiently differentmobility have already been resolved according to mobility by the knownmethods. Other ion mixtures contain ion species which are not resolvedaccording to mobility.

The invention provides a method for analyzing a substance mixture inwhich a first mass-mobility map is acquired using a mass spectrometerwhich comprises a trapped ion mobility separator and a time-of-flightanalyzer. The method is characterized by the fact that (a) an ion signalof interest is selected in the first mass-mobility map and examined asto whether it is a superimposition of signals from different ion specieswhich are not sufficiently resolved according to mass or mobility, andthat (b) a second mass-mobility map is acquired wherein at least oneparameter of the trapped ion mobility separator is changed such that theregion around the ion signal of interest is measured in the second mapwith a higher mobility resolution and/or mobility accuracy compared tothe first map.

An “ion signal” is understood to be the signal of those ions which arereleased almost simultaneously from the trapped ion mobility separatorduring the mobility scan and transferred to the downstream massanalyzer.

The second, more detailed mass-mobility map is also called a “zoom map”hereinafter; it no longer contains all ion signals, but only the ionsignals of the ion species selected in narrow mass and mobility ranges.

The invention provides a further method in which a first mass-mobilitymap is acquired using a mass spectrometer which comprises a trapped ionmobility separator, a mass separator, a fragmentation cell and atime-of-flight analyzer. Ion signals are selected from the firstmass-mobility map and then fragment ion spectra of ions of the selectedion signals are acquired, wherein the ions are selected according tomobility and mass. The method is characterized by the fact that, in afirst step, one of the selected ion signals, its fragment ion spectrumand/or the substance identification, is investigated as to whether thision signal of interest is a superposition of signals of different ionspecies, which are not sufficiently resolved according to mass ormobility. In a second step, a second mass-mobility map is acquiredwherein at least one parameter of the trapped ion mobility separator ischanged for the analysis of the ion signal of interest such that theregion around the ion signal of interest is measured in the second mapwith a higher mobility resolution and/or mobility accuracy compared tothe first map. In the second step, additionally or alternatively, anadditional fragment ion spectrum of ions of the ion signal of interestis acquired, wherein at least one parameter of the trapped ion mobilityseparator is changed such that the ions, selected according to mobilityand mass, of the additionally acquired fragment ion spectrum have asmaller mobility bandwidth ΔK compared to those of the fragment ionspectrum acquired before.

The mass-mobility map from which the mobility or the collisioncross-section and the mass of the ion signal are determined, and/or thefragment ion spectrum, can be used for the substance identification ofthe ion signals. The additional fragment ion spectrum is preferably usedfor the substance identification of the ion signal of interest. Themobility or the collision cross-section of the ion signal of interestcan additionally or alternatively be determined from the secondmass-mobility map and used for the substance identification of the ionsignal of interest.

The ion signal of interest can be analyzed with regard to the signalwidth or signal form, or both, as to whether it is a superimposition ofsignals of different ion species. The signal width and/or the signalform of the ion signal of interest are preferably compared with thesignal width or signal form of a single ion species and investigated inparticular for a deviation along the mobility direction.

The at least one parameter of the trapped ion mobility separator whichis changed in order to measure the region around the ion signal ofinterest with increased mobility resolution and/or increased mobilityaccuracy in the second map, is preferably the scan speed of the trappedion mobility separator, in particular a reduction in the scan speedduring the scan around the ion signal of interest. The scan speed of thetrapped ion mobility separator can be reduced there as well in order toselect ions with a smaller mobility bandwidth ΔK for the additionalfragment ion spectrum.

The ion signal of interest is selected from the ion signals of the firstmap, using a selection list containing known isomers or isomers ofparticular analytical interest. The selection list here is preferablydependent on the type of substance mixture. The selection can be done bya user as well, however, who marks the ion signal of interest in avisual representation of the first map. The extent of the marking in thedirection of mobility here can set the mobility resolution of the secondmap in the region of the ion signal of interest and specify theparameter(s) of the trapped ion mobility separator. It is preferablethat only those ion signals are selected which have not already beenselected and identified in a previous map.

The acquisition of the first and/or second map can involve a calibrationof the mobility axis so that the mobility or the collision cross-sectionof the ion signal of interest can be determined. For the calibration, itis preferable if the known mobilities or collision cross-sections ofidentified substances of the substance mixture or of referencesubstances are used, the reference substances being added to thesubstance mixture. In particular, at least two reference substanceswhose mobilities encompass the mobility of the ion signal of interestcan be added to the substance mixture.

The substance mixture can be fed to the ion source in dissolved formwith a syringe pump, with the mass-mobility maps remaining essentiallythe same from measurement to measurement.

The substance mixture can also be first separated in a chromatograph andfed to the ion source as a separated substance mixture, however. Thechromatography system is preferably a liquid chromatography (LC) systemand the separated substance mixture is fed to the ion source in liquidform. The chromatography system (substance separator) can also be a gaschromatography system or carry out an electrophoretic separation, e.g.in the form of a capillary electrophoresis. For complex mixtures, aplurality of substances can still be superimposed even after aseparation.

If the substances of the substance mixture are separated by means of asubstance separator and fed to the ion source, the ion signals belongingto a substance only appear in mass-mobility maps being acquired intemporal sequence when the concentration of the substance has exceeded asensitivity threshold. The concentration then increases initially infurther mass mobility maps, reaches a maximum and decreases again. It isparticularly favorable that an ion signal is only displayed in a visualrepresentation of a mass-mobility map, or an ion signal of interest isonly selected, when the ion signal has reached the maximum during thesubstance separation. Furthermore, an approximately constantconcentration of the substance exists for a short time near the maximum.

If several samples with similar composition are to be separated andanalyzed, for example always in the same way regarding the presence ofspecific isomers, then the optimum device parameters of the substanceseparator and/or the mass spectrometer can be determined for substancesof interest in one or more cycles of a first sample and used for thesamples subsequently analyzed. For a series of similar samples, themethod according to the invention is therefore worked out in detail withthe aid of a first sample and then used for the further samples in thesame way. It is therefore expedient to save the device parameters whichchange during the separation process of the first sample and to re-usethem later for further similar samples. At the known retention times ofspecific substances of interest, the corresponding device parameters ofthe mass spectrometer are used in particular, especially thecorresponding device parameters of the trapped ion mobility separator,the mass filter, the fragmentation cell and/or the time-of-flightanalyzer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic representation of a typical trapped ionmobility separator and an operating mode of the trapped ion mobilityseparator.

FIG. 2 shows the mobility resolution of a trapped ion mobility separatoras a function of the scan duration.

FIG. 3 shows a mass spectrometric system with which the methodsaccording to the invention can preferably be carried out. The use of thetrapped ion mobility separator (TIMS) with its adjustable mobilityresolution allows analyses to be undertaken on individual ion specieswith highest mobility resolution.

FIG. 4 shows a schematic representation of a mass-mobility map which isacquired with the mass spectrometric system shown in FIG. 3, without themass filter and the fragmentation cell being in operation. A group ofeight ion signals, whose ions can be identified in a subsequentmeasuring cycle, is selected in the map. FIG. 4 is only a roughschematic of a mass-mobility map; many details are not visible here. Notshown, for example, is the fact that an isotopic distribution can beseen for each ion signal due to the high mass resolution of thetime-of-flight mass spectrometer.

FIG. 5 shows a preferred operating mode of a trapped ion mobilityseparator with parallel accumulation.

FIG. 6 shows a schematic representation of a mass-mobility map in whicha series of ion signals (53) is resolved according to mobility. Furtherion signals (50, 51, 52) comprise several ion species, but are notresolved according to mobility. If these ion signals are of particularanalytical interest, they can be measured in a subsequent proceduralstep, resolved according to their mobility, particularly by usingzooming methods.

FIG. 7 shows a schematic representation of a second-mass mobility map(zoom map) of signal group (50) from FIG. 6, in which the mass range islimited to 15 daltons, and the mobility range of the signal group (50)is swept by a slow scan of 400 milliseconds. The signal group is nowresolved according to mobility and also exhibits its isotopic structurein this representation. Signal group (50 d) seems to be an isobar with adifferent isotopic composition.

FIG. 8 shows a schematic representation of a mass-mobility map, in whichan analyte signal of particular interest (55) is surrounded by twocalibration signals (54) and (56) of accurately known mobility in orderto determine the mobility of the analyte signal (55) accurately in azoom scan.

FIG. 9 shows a preferred operating mode of a trapped ion mobilityseparator with parallel accumulation, in which a “spatial zoom” is usedto analyze a limited mobility range and minimize the effect of the spacecharge.

FIG. 10 depicts a constructed section of an LC chromatogram from second900 to second 952, with several substance peaks which each have a fullwidth at half-maximum of 10 seconds. Line (60) represents thesensitivity threshold. If one mass-mobility map is measured everysecond, represented by the vertical broken lines, the substance drawn asa broken line first appears at time t=929 s with intensity (61). In thesubsequent mass mobility maps, the intensity increases from (62) to(68), with the intensity (68) lying at the start of the intensitymaximum. A program-controlled analysis can determine when the maximum isreached for every substance, and offer only those substances whichappear close to the maximum, and have not yet been identified inprevious measuring cycles, for selection of the substances to bemeasured, as is shown in FIG. 4.

DETAILED DESCRIPTION

FIG. 4 shows a schematic representation of a mass-mobility map which isacquired with the mass spectrometric system from FIG. 3, without themass filter and the fragmentation cell being in operation. A group ofeight ion signals, whose ions can be identified in a subsequentmeasuring cycle, is selected in the map. FIG. 4 is only a roughschematic of a mass-mobility map; many details are not visible here. Notshown, for example, is the fact that each ion signal exhibits anisotopic distribution due to the high mass resolution of thetime-of-flight mass spectrometer. In a visual representation of detailsof the map, an ion signal appears as a bunch of short horizontal lines,which can be recognized as bunches only at high screen zoom factor.

After the trapped ion mobility separator (TIMS cell) has been filledwith sufficient ions, a mobility scan is started to identify selectedion signals. When the first ion signal appears at the exit of thetrapped ion mobility separator, the mass filter is set to the mass ofthis ion signal and the ions are allowed to pass through the massfilter. The filtered ions are then fragmented in the fragmentation celland the fragment ions are measured in the time-of-flight mass analyzer.It is usually possible to determine the substance of the ion signal fromthe fragment ion spectrum. The acquisitions of the fragment ion spectraof the second, third substance and so on then follow. When all eight ionsignals of this signal group have been measured, the TIMS cell can berefilled and a second group of ion signals can be measured in a secondmeasuring cycle.

The invention relates to a method for the detailed analysis of organicsubstances in complex mixtures which is conducted in a mass spectrometercomprising an ion source, a trapped ion mobility separator (TIMS), amass separator, a fragmentation cell and a time-of-flight analyzer. Inprinciple, mobility spectrometers can separate both isomers and isobars.They can usually separate near isobars as well.

In a first cycle, at least one mass-mobility map is acquired andselected ion signals from these maps are identified by the acquisitionand evaluation of fragment ion spectra of the ions of these ion signals(first cycle).

The ion signals, their fragment ion spectra, their substanceidentifications and/or the identified substance structures are examinedas to whether ion signals from these maps are possibly superpositions ofdifferent ion species which are not sufficiently resolved according tomass or mobility, and whether this means a mixture of isomers, isobarsor near isobars is possibly present.

In a second cycle, further maps and/or fragment ion spectra areacquired, wherein detailed methods with changed method parameters areused in order to measure ion signals of analytical interest withincreased mobility resolution or mobility accuracy, in particular inorder to measure or select ions of analytical interest separately fromother ion species. Detailed methods of the trapped ion mobilityseparator are particularly the temporal or spatial zoom. The mobilityresolution or mobility separation selectivity around an ion signal ofanalytical interest can be higher than 100, preferably higher than 300,in particular higher than 500 here.

With many analytical methods it is not only important to detect groupswith several isomers. If it is known that several isomers can be presentfor one substance of a substance group, and if only one isomer is foundfor this substance, it can be of particular analytical interest whichisomer is present. For conformational isomers, in particular, thefragment ion spectra often only differ so slightly that a reliableidentification of the isomer with the aid of this spectrum is notpossible. If a complete group of isomers is found, then it is usuallypossible to determine the isomers by means of the structure of theisomeric group; this is usually not possible for an isomer that isindividually present, however. If a fragment ion analysis does not giveany indication of the identity of the isomer, the isomer can often beidentified via a precise determination of the mobility or the collisioncross-section.

FIG. 6 shows a schematic representation of a mass-mobility map in whicha series of ion signals (53) is resolved according to mobility. Furtherion signals (50, 51, 52) comprise a mixture of ions of several ionspecies, which are not resolved according to mobility, however. Thepresence of ion mixtures can often be detected via a temporally extendedion signal in the mobility direction, in particular if the map hasalready been acquired with a correspondingly high mobility resolution.The presence of ion mixtures often cannot be seen from the signal atall. If these ion signals are of particular analytical interest, theycan be measured in a subsequent procedural step, resolved according totheir mobility, particularly by using zooming methods.

The search for ion signals which are possibly unresolved signalsuperpositions of different ion species can be carried out in a varietyof ways:

(a) Computer programs can automatically analyze the ion signals in themass mobility maps, in particular their temporal width (signal width)and intensity distribution (signal form), and also overlaps of isotopicdistributions.

(b) A user can examine the ion signals visually, and can be supportedhere by databases, which contain in particular those substance groupswhich frequently contain isomers. The databases here can in particularcontain substance groups of particular analytical interest and bespecific to the type of substance mixture under investigation.(c) The fragment ion spectra should differ from each other, particularlyin the case of isobars and near isobars, and facilitate the recognitionof a mixture of different ion species. It is thus possible to identifyion mixtures with different numbers of double bonds.(d) Substances which are identified in the map of the first cycle canalso be examined as to the probability of the presence of isomers withthe aid of databases, even without an analysis of the ion signals. Hereas well, the databases can particularly contain substance groups, wherethe measurement of isomers is of particular analytical interest, or canbe specific to the type of substance mixture under investigation. Thismeans that ion signals whose signal forms and widths exhibit nopeculiarities can also be included in the measurement of a second cycle.Some substance groups have a tendency to form isomers whose mobilitiesdiffer only very slightly, for example because with two isomers, adouble bond can be located at two adjacent carbon atoms. Thesedistinctions can particularly be of pharmacological importance. If onlyone isomer is present, it is often of particular interest to determineaccurately which isomer it is.(e) The at least approximately measured collision cross-sections a canbe compared with library-type collections of substance data with theircollision cross-sections in an automated way by computer programs, forexample, and lead to more accurate measurements in the event ofdiscrepancies.

It should be noted here that it does not always make sense for all ionsignals which could contain isomers or isobars to be measured withincreased mobility resolution in the second cycle also. Priority hereshould be given to the analytical significance.

It is not necessarily always only isomers or isobars which lead to ionsignals which are a superposition of ion signals of different ionspecies and appear without being cleanly resolved according to mass andmobility. The mass filter is usually set such that the whole isotopicgroup is transmitted for one ion species; for heavier ions, thetransmission width can optimally be around five daltons or more. Thismeans that mixtures of ion species whose molecular masses differ by onlya few daltons are no longer cleanly separated, however. If the moleculesare very different, the mixture can be identified from the fragment ionspectra. They can also have nearly the same structure, however, forexample when they have slightly different numbers of double bonds. Theirmolecular masses then each differ only by two, four or six daltons (forsingly charged ions). Those mixtures of ion species with molecularmasses which differ by only a few daltons can be separated by the methodaccording to the invention in a second cycle with a correspondinglyincreased mobility resolution.

If ion signals are found for which a particular interest in a detailedanalysis exists, the identification method can be performed a secondtime with the same substance mixture, but for each ion signal ofinterest, detailed methods are specifically set-up with specificallydetermined method parameters.

A temporal zoom is usually set here, which expands the mobility scan forthe ion signals of analytical interest such that an enhanced mobilityresolving power is achieved around these ion signals (see U.S. Pat. No.8,766,176 B2; “Acquisition Modes for Ion Mobility Spectrometers usingTrapped Ions”, D. A. Kaplan et al.). How high the resolution has to becan often only be specified by experience or from information insuitable libraries. It is preferable if mobility resolutions ofR_(mob)=150 to 250 are set.

If the ion signals can be superpositions, and if these ion signals areof sufficiently high analytical interest, then, after a renewed fillingof the TIMS cell, further maps and/or fragment ion spectra are acquired,for which detailed methods with changed method parameters are used inorder to measure the ion signals of analytical interest with increasedmobility resolution or mobility accuracy, in particular in order tomeasure or select ions of analytical interest separately from other ionspecies The mobility resolution can be higher than 100, higher than 300,or even higher than 500, depending on the requirement. This second massmobility map with increased mobility resolution will be called “zoommap” hereinafter.

Such a zoom map is shown in FIG. 7, albeit in a greatly simplified form.All intensities of the ions of every individual isotopic distributionare drawn identically here in order to make the difference between theisotopic distributions (50 c) and (50 d) visible. The zoom map depictsthe signal group (50) of FIG. 6. It shows that the signal group (50)comprises four individual ion species, of which the fourth ion species(50 d) is probably an isobar, since a different type of isotopicdistribution is present. The zoom map shows only a mass range of 15daltons and the mobility axis is expanded to 400 milliseconds by atemporal mobility zoom in order to achieve the high mobility resolution.

Several ion signals, which now usually represent individual ion species,i.e. isomers, isobars or near isobars, can then be selected again fromthe zoom map. In a further step of the method, fragment ion spectra ofthese ion signals can be acquired. The fragment ion spectra can be usedfor the identification of the now separated isomers, isobars or nearisobars, although an unequivocal identification is not always achieved,because the fragment ion spectra of isomers can be very similar. If itis known that several isomers can be present for one substance of asubstance group, and if only one isomer is found for this substance, itcan be of particular analytical interest which isomer is present.

A temporal zoom can also be supported by a spatial zoom. FIG. 9illustrates a preferred operating mode of a trapped ion mobilityseparator with parallel accumulation, in which a “spatial zoom” is usedto analyze a limited mobility region and minimize the effect of thespace charge and thus losses due to the space charge. For a spatialzoom, a ramp is set up in the storing part (11 a) as well as in thescanning part (11 b) by additional voltages at the electrodes of thetube, where a part of the ramp between positions (41) and (42) andbetween (45) and (46) respectively is kept very flat in order to storeions of a very small mobility range at large separations from eachother. Many more ions can thus be stored in this mobility range, sincethe influence of the space charge on the ions is suppressed. This methodis described in detail in U.S. patent application Ser. No. 14/931,125(M. A. Park and O. Raether; “Spatial Zoom Mode for Accumulative TrappedIon Mobility Spectrometry”).

The determination of the optimal method parameters for the ion signalsof analytical interest can be done automatically, for example in thecase when the ion signal reveals a superposition structure in a massmobility map of the first cycle. The mobility resolutions required forthe ion signals of analytical interest can also be taken from librariesof isomers, if available. The ion signals of analytical interest can benoted in a list of the ion signals which are obtained from the firstcycle. It is also possible to select the ion signals of interest simplyin a representation of the mass-mobility map on a computer screen bymarking the ion signals there, e.g. framed with a box. The size of thebox can also specify the desired resolution here.

If an accurate determination of the mobility K₀ or the collisioncross-section a of a substance is of interest, reference substances(calibration substances) of accurately known mobility (or accuratelyknown collision cross-sections) with similar or equal mass and onlyslightly different mobility can be added to the substance mixtureespecially for the second (or any further) cycle of the method. FIG. 8shows a schematic representation of a mass-mobility map, in which ananalyte signal of particular interest (55) is surrounded by twocalibration signals (54) and (56) of accurately known mobility in orderto determine the mobility of the analyte signal (55) accurately in asubsequent cycle with a temporal zoom. It is preferable to add at leasttwo reference substances to both sides of the ion signal of analyticalinterest, if possible, so that the mobility of the ion signal and thatof the two reference substances can be measured in a temporal zoom of amobility scan. The mobility of the substance can then be interpolatedwith high accuracy and precision from this measurement. For a calibratedmeasurement, the known mobilities or collision cross-sections of alreadyidentified substances of the complex substance mixture can also be used,however.

The ion source depicted in FIG. 3 is preferably an electrospray ionsource (ESI), to which the substances are fed in liquid form; otherionization types can also be used, however, such as thermosprayionization, chemical ionization or photoionization, each at atmosphericpressure after vaporization of the liquid which has been fed in, orLiquid Injection Field Desorption Ionization (LIFDI). The substances canbe fed to the ion source in gaseous form as well, however, e.g. bycoupling to a gas chromatograph, wherein the ionization can take placewith the aid of electron impact ionization or chemical ionization, forexample.

The substances can also be fed directly to the ion source as dissolvedsubstances by a syringe pump or after separation by means of liquidchromatography or electrophoresis. A chromatographic pre-separation cantake an hour or longer and provide a plurality of mass mobility mapswith a few hundred thousand ion signals.

If the substances of the complex mixture are fed in after separation bychromatography or electrophoresis, the ion signals belonging to asubstance appear in the mass-mobility maps acquired roughly once asecond only when the concentration of the substance has exceeded asensitivity threshold. FIG. 10 depicts a constructed section of an LCchromatogram from second 900 to second 952, with substance peaks whicheach have a full width at half-maximum of 10 seconds. Line (60)represents the sensitivity threshold. If one mass-mobility map ismeasured every second, represented by the vertical broken lines, the ionsignal for the substance drawn as a broken line appears for the firsttime at time t=929 s and with intensity (61). In the subsequentmass-mobility maps, the intensity of this ion signal increases from (62)to (68), with the intensity (68) lying at the start of the intensitymaximum. A program-controlled analysis can determine for each ion signalof the mass-mobility maps when the maximum is reached. It is thenpossible to only offer substances near the maximum for the selection ofthe ion signals whose fragment ion spectra are to be measured (see FIG.4). The same also applies to ion signals which are to be subjected to adetailed analysis for isomers, isobars, or near isobars.

FIG. 4 can be construed in a completely new way when this procedure oflimiting to maxima is applied: The ion signals of the mass-mobility mapshould only be those ion signals which are near the maxima of the LCpeaks and which have not already been identified in earlier measuringcycles. This method helps to make the mass mobility maps for extremelycomplex substance mixtures more clear.

In the case of a pre-separation by means of liquid chromatography orelectrophoresis, a calibration is difficult, since calibrationsubstances usually elute at different times to the analyte substances. Acalibration solution can be added, however, ideally in an automated way.

It should be noted here that series of analyses are often conducted onsubstance mixtures which are known to have nearly the same compositionand are all subject to the same analytical objective. If a detailedmethod (second cycle) with satisfactory fine analyses of isomers,isobars or near isobars has been established for this type of substancemixture, it is not absolutely necessary to carry out the first cycle. Afirst cycle carried out once can be used as the basis for analyses of aspecific type of substance mixture. If large numbers of samples with asimilar type of composition are to be analyzed with the aid of achromatographic or an electrophoretic separation, for example always inthe same way for the presence of specific isomers, the detailed methodsobtained and developed in first separation cycles of a first sample canrun automatically in separations of further samples, whereby theparameters belonging to the given retention times are used. The proposalis therefore to save the parameter changes during the cycles and tore-use them later for similar samples.

If the mass spectrometer is equipped with computers with sufficientcapacity and sufficient speed, and if corresponding programs can beprovided, then the fully automatic measurement of mass-mobility maps,selection of groups of ion signals, measurement of the fragment ionspectra, selection of ion signals of particular analytical interest,measurement of zoom maps, and measurement of the fragment ion spectra ofthe now separated ion species in a single LC run is possible. If the ionsignals of interest are selected with the aid of the user, then at leasttwo LC runs are required, with the zoom maps being measured in thesecond LC run.

The invention claimed is:
 1. A method for analyzing a substance mixturein a mass spectrometer which comprises a trapped ion mobility separator,a mass separator, a fragmentation cell, and a time-of-flight analyzer,wherein a first mass-mobility map is acquired, ion signals from thefirst mass-mobility map are selected, and subsequently fragment ionspectra of ions of the selected ion signals are acquired, said ionsbeing selected according to mobility and mass, further comprising thesteps: (a) investigating one of the selected ion signals, its fragmention spectrum and/or the substance identification as to whether this ionsignal of interest is a superposition of signals of different ionspecies which are not sufficiently resolved according to mass ormobility, and (b) acquiring a second mass-mobility map wherein at leastone parameter of the trapped ion mobility separator is changed suchthat, in the second map, the region around the ion signal of interest ismeasured with a higher mobility resolution and/or higher mobilityaccuracy compared to the first map, and/or acquiring an additionalfragment ion spectrum of ions of the ion signal of interest wherein atleast one parameter of the trapped ion mobility separator is changedsuch that the ions, selected according to mobility and mass, of theadditionally acquired fragment ion spectrum have a smaller mobilitybandwidth ΔK compared to the ions of the fragment ion spectrum acquiredbefore.
 2. The method according to claim 1, wherein the additionalfragment ion spectrum is used for the substance identification of theion signal of interest.
 3. The method according to claim 1, wherein themobility or collision cross-section is used for the substanceidentification of the ion signal of interest.
 4. The method according toclaim 1, wherein the signal width or signal form, or both, of the ionsignal of interest is examined as to whether it is a superposition ofsignals of different ion species.
 5. The method according to claim 4,wherein the signal width or the signal form or both are examinedregarding a deviation from the signal width or signal form of a singleion species.
 6. The method according to claim 5, wherein a deviationbetween the ion signal of interest and the ion signal of a single ionspecies along the mobility direction is analyzed.
 7. The methodaccording to claim 1, wherein the at least one parameter of the trappedion mobility separator is the scan speed in the region of the ion signalof interest.
 8. The method according to claim 1, wherein a selectionlist which contains known isomers or isomers of particular analyticalinterest is used for the selection of the ion signal of interest fromthe ion signals of the first map.
 9. The method according to claim 8,wherein the selection list depends on the type of substance mixture. 10.The method according to claim 1, wherein a user marks the ion signal ofinterest in a visual representation of the first map.
 11. The methodaccording to claim 10, wherein the extent of the marking in thedirection of the mobility specifies the mobility resolution of thesecond map in the region of the ion signal of interest and determinesthe at least one parameter of the trapped ion mobility separator. 12.The method according to claim 1, wherein the mobility axis of the firstand/or second map is calibrated and the mobility or collisioncross-section of the ion signal of interest is determined.
 13. Themethod according to claim 12, wherein the known mobilities or collisioncross-sections of identified substances of the substance mixture areused for the calibration.
 14. The method according to claim 12, whereinthe known mobilities or collision cross-sections of reference substanceswhich are added to the substance mixture are used for the calibration.15. The method according to claim 14, wherein at least two referencesubstances whose mobilities encompass the mobility of the ion signal ofinterest are added to the substance mixture.
 16. The method according toclaim 12, wherein the mobility or collision cross-section is used toidentify the substance components of the ion signal of interest.
 17. Themethod according to claim 1, wherein the samples are separated by asubstance separator, in particular a liquid chromatograph, and fed tothe mass spectrometer.
 18. The method according to claim 17, wherein anion signal is only displayed in a visual representation of amass-mobility map, or an ion signal of interest is only selected whenthe ion signal has reached the maximum during the substance separation.19. The method according to claim 1, wherein the method is worked out indetail with the aid of a first sample for a series of similar samplesand is then used for the further samples in the same way.